Abstract. The focus of this proposal is the development of therapy to protect the lung with an extracellular anti-oxidant defense to treat chronic obstructive pulmonary disease (COPD), a chronic disorder in which inhaled oxidants from tobacco smoke, pollution and generated within the lung by activated inflammatory cells perpetuate injury to the lung epithelium and endothelium. The LEXEO strategy is to use in vivo gene O2? therapy technology to provide a persistent extracellular anti-oxidant enzyme shield to the lung that will inactivate superoxide ( ) and H2O2, major extracellular oxidant stresses to the lung. LEXEO will capitalize on an innovative strategy developed by the Crystal laboratory at Weill Cornell, in which genes for catalase and superoxide dismutase 3 (SOD3) have been genetically modified to secrete functional monomeric antioxidant enzymes that can diffuse in the extracellular milieu, providing the lung with an effective extracellular anti-oxidant shield. Catalase is a tetrameric intracellular enzyme that is too large to diffuse if designed to be secreted. To use catalase as an effective extracellular anti-oxidant, the gene was modified to prevent the wrapping loop domain to mediate tetramer formation. With the addition of a secretory signal, the human catalase monomer (hCatWL?) is secreted, capable of functioning to catalyze extracellular H2O2 to H2O. Superoxide dismutase 3 (SOD3) is secreted, but it is a large tetramer, and has a heparin-binding domain that attaches it to cell surfaces. To modify SOD3 into a more effective lung extracellular antioxidant, a loop critical for tetramer formation was modified and the heparin-binding domain removed (hSOD3hd?), resulting in an effective monomer antioxidant enzyme that will not bind to cell surfaces. A serotype rh.10 adeno-associated (AAV) gene transfer vector will be used to genetically modify the liver to express and secrete the modified catalase and/or SOD3 monomers. Identification of the optimal vector for further clinical development in a phase 2 future proposal will be carried out in 2 steps: in vitro (aim 1) and in vivo. Four AAVrh.10 candidates will be evaluated: AAVrh.10hCatWL? (expressing the catalase monomer); AAVrh.10hSOD3hd? (SOD3 monomer); AAVrh.10hCatWL?/hSOD3hd? (both monomers); and AAVrh.10hSODhd?/hCatWL? (same but with SOD3hd? in the 5? position). The deliverable is to identify LEX05, the best candidate AAVrh.10 vector that will generate a persistent extracellular antioxidant shield of the lung. Aim 1. To compare in vitro the levels of expression of secreted, functional modified catalase and/or SOD3 mediated by AAVrh.10 vectors with the expression cassettes (hCatWL?, hSOD3hd?, hCatWL?/hSOD3hd? and hSOD3hd?/hCatWL?). Aim 2. In an in vivo murine model of COPD, quantify the ability of the optimal antioxidant vector from aim 1 to express secreted, functional modified catalase and SOD3 capable of protecting lung endothelium and epithelium from chronic exposure to cigarette smoke.